Abstract
Background: Our previous work showed that pharmacological blockade of Osteopontin (OPN) signaling can prevent and reverse heart failure induced by pressure overload in a transverse aortic construction (TAC) mouse model. Surprisingly, OPN Knockout (KO) mice subjected to 3 month TAC had worse cardiac function and bigger hearts than wild type (WT) TAC mice, despite lack of cardiomyocyte hypertrophy. We hypothesized that OPN KO increased adult cardiomyocyte proliferation in TAC-induced heart failure. Methods: Male C57Bl/6 (n=17) or OPN KO (n=11) mice were subject to TAC for 3 months. The protein levels of the mitosis marker H3P was quantified using immunofluorescence in paraffin-embedded myocardial sections. Myocytes were co-stained with WGA and MLC2 to count the number of myocytes per field. Adult primary cardiomyocytes from WT hearts were isolated and analyzed with co-imunoprecipitation (Co-IP) to study the interation between the regeneration factor YAP1, OPN and OPN-regulated proteins such as LDLR. For validation and mechanistic studies, more Co-IP experiments were performed in proliferative human liver HEPG2 cells. To study the effect of OPN blockade on YAP1 nuclear translocation, HEPG2 cells and human iPSC- derived cardiomyocytes (hiPS-CMs) were treated with an IgG or OPN blocking antibody for 24 hours followed by immunostaining for YAP1 and PITX2. Results: Nuclear H3P normalized to myocyte count was significantly increased in OPN KO relative to WT TAC hearts (Fold Change = 1.4; p=0,04). Co-IP results revealed a novel interaction between OPN, LDLR and YAP1. Stimulation of β2 adrenergic receptor increased the formation of this multi-molecular complex in a time-dependent manner. Blockade of OPN by a monocolonal antibody for 24 hours caused nuclear localization of YAP1 and PITX2 in HEPG2 cells and hiPS-CMs. Conclusion: OPN regulates the mitotic program in adult cardiomyocytes. Furthermore, the interaction of OPN with LDLR and YAP1 to form a new multi-molecular protein complex is regulated by β2-cAMP signaling pathway. Importantly, OPN regulates nuclear translocation of the regeneration factors YAP1 and PITX2, suggesting that OPN signaling may be important for adult cardiomyocyte division in TAC and potentially myocardial infarction and aging.